A Mitogen-activated protein kinase controls differentiation of bloodstream forms of Trypanosoma brucei

Abstract

African trypanosomes undergo differentiation in order to adapt to the mammalian host and the tsetse fly vector. To characterize the role of a mitogen-activated protein (MAP) kinase homologue, TbMAPK5, in the differentiation of Trypanosoma brucei, we constructed a knockout in procyclic (insect) forms from a differentiation-competent (pleomorphic) stock. Two independent knockout clones proliferated normally in culture and were not essential for other life cycle stages in the fly. They were also able to infect immunosuppressed mice, but the peak parasitemia was 16-fold lower than that of the wild type. Differentiation of the proliferating long slender to the nonproliferating short stumpy bloodstream form is triggered by an autocrine factor, stumpy induction factor (SIF). The knockout differentiated prematurely in mice and in culture, suggestive of increased sensitivity to SIF. In contrast, a null mutant of a cell line refractory to SIF was able to proliferate normally. The differentiation phenotype was partially rescued by complementation with wild-type TbMAPK5 but exacerbated by introduction of a nonactivatable mutant form. Our results indicate a regulatory function for TbMAPK5 in the differentiation of bloodstream forms of T. brucei that might be exploitable as a target for chemotherapy against human sleeping sickness.

FIG. 1.

TbMAPK5 is related to MAP kinases. (A) The amino acid sequence of TbMAPK5 is displayed by Clustalx1.82 alignment (37) with MAP kinases from Dictyostelium discoideum (ERK1_DICDI, EMBL accession no. P42525) (13), Rattus norvegicus (ERK1_rat, EMBL accession no. P21708) (22), Nicotiana tabacum (MAPK_Nt, EMBL accession no. U94192.1) (48), and L. mexicana (MPK5_Lm, EMBL accession no. AJ93283) (47). Identical and conserved amino acids are shaded black and gray, respectively. The start and the end of the catalytic domain are indicated by vertical arrows, sequences highly diagnostic for MAP kinases (8, 19) are indicated by black diamonds, and conserved amino acids of the common docking domain are indicated by spades. Domains with known functions, including the ATP binding site and a domain diagnostic for serine/threonine kinases (S/T), are overlined. (B) Radial phylogenetic tree of the MAP kinases described above, together with MPK3 (MP_114433) from Arabidopsis thaliana, KSS1 (M26398) from yeast, ERK5 (Q13164) and JNK2α1 (AAC50606) from humans, SAPKα (P92208) from Drosophila melanogaster, p38 (Q9Z1B7) from mice, KFR1 (18) and MAPK2 (26) from T. brucei, and MAPK1 (NC_702183) and MAPK2 (JC5153) from Plasmodium falciparum were constructed with the TreeView software (http://taxonomy.zoology.gla.ac.uk/rod/rod.html). Bootstrap values indicating the number of occurrences of branch points during 1,000 replications are given for the three primary branches consisting of the ERK subgroup, stress-activated protein kinases, and MAP kinases from protozoa.

FIG. 2.

Nucleic acid analyses of TbMAPK5. (A) Steady-state level of TbMAPK2 mRNA. Equal amounts of total RNA (10 μg) extracted from long slender (LS) and short stumpy (SS) bloodstream forms (BSF), synchronously differentiating forms 2 h and 6 h after triggering of differentiation, and early and late procyclic forms (PCF) cultured in the presence or absence of glycerol were loaded. Expression was analyzed by Northern blotting with a radiolabeled, TbMAPK5-specific probe (top). Hybridization signals were quantified with a PhosphorImager and normalized to the 18S rRNA (bottom). The values below indicate the relative amounts of TbMAPK5-specific mRNA in the different stages. nt, nucleotides. (B) Southern blot analysis of a TbMAPK5 null mutant of monomorphic clone MITat 1.2. Genomic DNA was digested with BamHI and hybridized with digoxigenin-labeled probes from the ORF of TbMAPK5 (top) or the phleomycin resistance gene (phleo, bottom).

FIG. 3.

The TbMAPK5 null mutant of monomorphic clone MITat 1.2 is able to grow normally. (A) Population growth of bloodstream form trypanosomes of the wild type (MAPK5/MAPK5), the heterozygous mutant (MAPK5/Δmapk5::HYG), and the null mutant(Δmapk5::HYG/Δmapk5::BLE). (B) Population growth upon triggering of differentiation (with cis-aconitate) of the same clones as above. Cells were diluted at daily intervals to ensure logarithmic growth. Population growth was calculated as cell density multiplied by the cumulative dilution factors.

FIG. 4.

Analysis of TbMAPK5 null mutants and an add-back mutant of pleomorphic clone AnTat 1.1. (A) Southern blot analysis of the wild type (wt) and two independent TbMAPK5 null mutant clones. Genomic DNA was digested with BamHI and hybridized with digoxigenin-labeled probes from the ORF of TbMAPK5, the hygromycin resistance gene (hygro), or the phleomycin resistance gene (phleo). (B) Northern blot analysis. Total RNA was extracted from bloodstream form trypanosomes of the wild type, the null mutant, and the add-back mutant and hybridized with radiolabeled sequences from TbMAPK5 (top) or, as a control for sample loading, with an 18S rRNA-specific probe (bottom). Hybridization signals were quantified with a phosphorimager and normalized to the 18S rRNA. TbMAPK5-specific hybridization signals of the wild type (2.7 kb) and the add-back mutant (1.8 kb) are indicated by arrows. Values below the panel indicate relative amounts of TbMAPK5-specific mRNA. nt, nucleotides.

FIG. 5.

Course of parasitemia of the wild type, null mutant, and add-back mutant of AnTat 1.1 during chronic infections of immunocompetent mice. Mice were injected intraperitoneally with 1 × 10⁵ long slender bloodstream forms, and the ensuing parasitemia was monitored from tail blood. The mean ± the standard deviation of three mice is presented.

FIG. 6.

Course of chronic infections of immunocompromised mice. Mice were treated with cyclophosphamide 24 h prior to infection with 1 × 10⁵ trypanosomes. The mean ± the standard deviation of four mice is presented. Cell numbers below the detection limit (indicated by arrows) were set to 5 × 10⁶ cells/ml.

FIG. 7.

Null mutant cells undergo differentiation at a reduced parasite density. (A) Percentage of diaphorase-positive cells during the time course of chronic infections of immunocompromised mice. Two independent experiments are shown. Figures 6 and 7 are from the same set of experiments. (B) Diaphorase activity of the wild type and the null mutant harvested 3 days or 7 to 8 days postinfection of immunocompromised mice. Arrows indicate diaphorase-positive cells (intermediate and stumpy forms). At least 100 cells were counted per sample. (C) Synchronous differentiation of stumpy forms of the null mutant. Stationary-phase bloodstream forms were harvested from the blood of infected mice 4 days postinfection and triggered to differentiate to the procyclic form. The appearance of EP procyclin on the surface of trypanosomes was monitored by flow cytometry with monoclonal antibody TRBP1/247 (30).

FIG. 8.

The null mutant differentiates at low cell density in vitro. Bloodstream forms of the wild type, null mutant, and add-back mutant were harvested from mouse blood 3 days postinfection and subjected to in vitro culture in the presence or absence of conditioned medium from a bloodstream form culture of monomorphic line MITat 1.2. After 0 h, 24 h, and 48 h, aliquots were removed from the cultures and analyzed for cell morphology and diaphorase activity. At least 40 cells of the population harvested from mouse blood (0 h) and at least 100 cells of the population that had been cultured for 24 or 48 h, respectively, were counted per sample. The percentages of long slender (diaphorase negative, slender morphology), intermediate (diaphorase positive, slender or intermediate morphology), and stumpy forms (diaphorase positive, stumpy morphology) are indicated.